Culture medium composition useful for induction and proliferation of Taxus calli

Abstract

Disclosed is a culture composition containing micro and macro-ingredients, vitamins and other supplements such as phytagel, sucrose, picloram. The medium is useful in the formation and development of callus lines of Taxus sp., and resulting in production of taxol in high amounts.

We claim:  
     
       1. A culture medium for induction and proliferation of callus lines from explants, the culture medium comprising 
       
         
           
                 
                 
               
                     
                 
                   A) Macronutrients 
                     
                 
                   in the amounts of: 
                 
                   KNO 3   
                   about 2528 mg · L −1   
                 
                   MgSO 4 .7H 2 O 
                   about 246 mg · L −1   
                 
                   (NH 4 ) 2 SO 4   
                   about 134 mg · L −1   
                 
                   NaH 2 PO 4   
                   about 150 mg · L −1   
                 
                   CaCl 2 .2H 2 O 
                   about 150 mg · L −1   
                 
                   Na 2 -EDTA 
                   about 37.2 mg · L −1   
                 
                   FeSO 4 .7H 2 O 
                   about 27.8 mg · L −1 ; 
                 
                   B) Micronutrients in 
                 
                   the amount of: 
                 
                   H 3 BO 3   
                   about 3.0 mg · L −1   
                 
                   CoCl 2 .6H 2 O 
                   about 0.025 mg · L −1   
                 
                   CuSO 4 .5H 2 O 
                   about 0.025 mg · L −1   
                 
                   MnSO 4 .H 2 O 
                   about 10.0 mg · L −1   
                 
                   ZnSO 4 .7H 2 O 
                   about 2.0 mg · L −1   
                 
                   KI 
                   about 0.75 mg · L −1   
                 
                   Na 2 MoO 4 ,.2H 2 O 
                   about 0.25 mg · L −1 ; 
                 
                   C) Vitamins in the amounts of: 
                 
                   Myo-inositol 
                   about 100 mg · L −1   
                 
                   Nicotinic acid 
                   about 1.0 mg · L −1   
                 
                   Pyridoxin.HCl 
                   about 1.0 mg · L −1   
                 
                   Thiamine.HCl 
                   about 10.0 mg · L −1 ; and  
                 
                   D) Other Ingredients 
                 
                   in the amounts of: 
                 
                   Picloram 
                   about 0.1-about 20.0 mg · L −1   
                 
                   Sucrose 
                   about 10,000-about 40,000 mg · L −1   
                 
                   Phytagel 
                   about 2.0-about 4.0 mg · L −1   
                 
                   Antioxidants 
                   about 1.0-about 5.0 mg · L −1 . 
                 
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
       2. The medium as claimed in  claim 1 , wherein the ex-plants are selected from the group consisting of stem, needle, embryo and endosperm. 
     
     
       3. The medium as claimed in  claim 1 , wherein the antioxidants are selected from the group consisting of activated charcoal, PVP, ascorbic acid and mixtures thereof. 
     
     
       4. A method for culturing and producing Taxus plants, said method comprising the steps of inoculating any plant part of Taxus sp. in the the culture medium as claimed in  claim 1 , for a period of about 30 to about 60 days in dark and at a temperature ranging between about 24-about 26° C., to obtain the undifferentiated mass of callus culture, culturing the calli in conventional media to obtain rooted plants and culturing the rooted plants in the field. 
     
     
       5. A process for the enhanced production of taxol and/or taxane class of molecules in callus cultures which comprises: inoculating any plant part of the Himalayan Yew in the culture medium as defined in  claim 1  for a period of about 30 to about 60 days in dark and at a temperature ranging between about 24-about 26° C., to obtain the undifferentiated mass of callus, extracting the product by conventional solvent extraction methods and analyzing the compounds by HPLC for detection of Taxol, 10-DAB or other taxanes.